Kappa opioid receptor effectors and uses thereof

ABSTRACT

The present invention is a selective kappa opioid receptor effector, or a pharmaceutically acceptable salt thereof, useful for treating ethanol use disorder withdrawal, anxiety and/or depression, schizophrenia, mania or post-traumatic stress disorder.

INTRODUCTION

This application claims the benefit of priority of U.S. ProvisionalApplication Nos. 61/534,938, filed Sep. 15, 2011, the content of whichis incorporated herein by reference in its entirety.

This invention was made with government support under Grant Nos. R01DA031927 and U54 HG005031 awarded by the National Institutes of Health.The government has certain rights in the invention.

BACKGROUND OF THE INVENTION

For normal activities that produce rewards, there is a rapid habituationof the circuits involved and the behaviors will wane. However, foraddictive drugs habituation does not occur and dopamine release persistsdespite repetitive trials. Upon withdrawal of the drug, a decrease ofdopamine levels in the nucleus accumbens results, and this has beenobserved for opioids, cannabinoids, alcohol, amphetamines, and nicotine(Cami & Farre (2003) N. Engl. J. Med. 349:975). This loss of dopamineaccounts for the withdrawal syndromes observed with these drugs. Theprototype opioid drug is morphine. It produces many effects typical ofmost opioids including analgesia, euphoria, nausea, and respiratorydepression. Repeated use of opioids produces physical dependence andtolerance. These manifestations of opioid use are due to the threerecognized types of opioid receptors that are members of the GPCRfamily, the mu (μ), delta (δ), and kappa (κ) subtype receptors. Whilestimulation of the mu and delta receptors increases dopamine release inthe nucleus accumbens, κ opioid (KOP) receptor activation by itsendogenous ligand dynorphin-A reduces extracellular dopamine. It hasbeen suggested that stimulation of KOP receptor by endogenous opioidslike dynorphins will produce an aversive state and thereby counter theeffects of rewarding and addictive compounds like alcohol, cocaine andnicotine. Moreover, exogenous KOR agonists have also been observed toattenuate drug-taking behavior (Prisinzano, et al. (2005) AAPS J.7:E592; Xuei, et al. (2006) Mol. Psychiatry 11:1016; Hasebe, et al.(2004) Ann. NY Acad. Sci. 1025:404; Metcalf & Coop (2005) AAPS J.7:E704). However, it may be difficult to strike a balance betweenopposing the sense of reward gained by drugs of abuse and producing anaversive state; therefore, activation of the KOP receptor may not betherapeutically preferable. Although these statements appear contrary,KOP receptor agonists can both alleviate drug self-administration inanimal models (most likely via dopamine regulation) and also triggerrelapse. This conflicting dual action of KOP receptor agonists alludesto the complex physiological role of KOP receptors and underscores theneed for a variety of chemical tools to facilitate their furtherinvestigation.

Intracranial self-stimulation has become a useful means of assessingreward thresholds in rodents and nonhuman primates. In essence, ananimal will press a lever to electrically stimulate the brain viaimplanted probes. This “self stimulation” will be performed to a certainextent in training and that extent is an indication of the animal's“reward threshold.” Administration of “drugs of abuse” has been shown todecrease this reward threshold such that the animal will seek lessstimulation to achieve the desired effect. This model paradigm has beenlikened to positive hedonic states produced by drugs of abuse in humanaddicts. In rodents, the direct activation of KOP receptor usingselective agonists increases reward thresholds (mimicking the withdrawalstate) and creating a “depressive-like” state (where more selfstimulation is required to achieve the desired effect). Treatment withantagonists has been shown to restore reward thresholds in this model(Glick, et al. (1995) Brain Res. 681:147; Bruijnzeel (2009) Brain Res.Rev. 62:127). The restoration of reward thresholds may be a veryimportant step in drug abuse treatment as drug cessation is stronglynegatively reinforced by aversive feelings, which may be due to anincreased reward threshold. Therefore, the development of KOP receptorantagonists would be particularly beneficial in “resetting” thisthreshold. Furthermore, since an increased reward threshold may manifestas a “depressive state,” then KOP receptor antagonists can also bebeneficial for the treatment of depressive disorders.

There are molecules known to activate or inhibit the KOP receptor,including salvinorin A, ketazocine, U-50,488, 5′-guanidinonaltrindoleand JDTic((3R)-7-Hydroxy-N-((1S)-1-[[(3R,4R)-4-(3-hydroxyphenyl)-3,4-dimethyl-1-piperidinyl]methyl]-2-methylpropyl)-1,2,3,4-tetrahydro-3-isoquinolinecarboxamide).Many of these molecules are either direct derivatives of opium alkaloidssuch as GNTI(5′-guanidinyl-17-(cyclopropylmethyl)-6,7-dehydro-4,5alpha-epoxy-3,14-dihydroxy-6,7-2′,3′-indolomorphinan;Jones, et al. (1998) J. Med. Chem. 41:4911) or contain structuralelements borrowed from these alkaloids, as can be observed for JDTic(Thomas, et al. (2001) J. Med. Chem. 44:2687-2690) and ketazocine (Merz& Stockhaus (1979) J. Med. Chem. 22:1475-1483). One consequence of thislegacy is that many of the established potent and selective moleculesare structurally complex, containing multiple stereocenters andrequiring lengthy synthetic routes to construct modified analogues. Thenatural product Salvinorin A (Roth, et al. (2002) Proc. Natl. Acad. Sci.U.S.A. 99:11934-11939) is unique as a potent, non-nitrogenous KOPreceptor ligand. While not an alkaloid, Salvinorin A is equal instructural complexity to any of the isolated opiates. Even the widelyutilized, simplified agonist compound, U-50,488 (VonVoigtlander &Szmuszkovicz (1982) J. Med. Chem. 25:1125-1126) contains two chiralcenters.

Currently, there are currently no approved agents or compounds fortreating the altered reward pathways associated with drug addiction(Prisinzano (2005) supra). Accordingly, there is a need in the art foreffector chemotypes (possessing novel patterns of binding toward the KOPreceptor) that can be readily synthesized for use in analyzing the KOPreceptor as well as in therapeutic methods.

SUMMARY OF THE INVENTION

The present invention is a pharmaceutical composition containing aneffective amount of a kappa opioid receptor antagonist, or apharmaceutically acceptable salt thereof, and a physiologicallyacceptable carrier, wherein the kappa opioid receptor antagonist is acompound of Formula I:

wherein

X, Y and Z are independently selected from H,H; O; S or NH;

R¹ is H, a halogen group or a substituted or unsubstituted lower alkylor alkoxy group; and

R² is present or absent, and when present is a substituent on one ormore ring atoms and is for each ring atom independently H, a halogengroup, or a substituted or unsubstituted lower alkyl or alkoxy group. Insome embodiments, the pharmaceutical composition further includes akappa opioid receptor agonist.

The present invention also provides a pharmaceutical compositioncontaining an effective amount of a kappa opioid receptor agonist, or apharmaceutically acceptable salt thereof, and a physiologicallyacceptable carrier, wherein the kappa opioid receptor agonist is acompound of Formula II:

wherein

X′ is hydrogen or Br;

each Z′ is independently C, CH or N;

n is 0 or 1;

R³ is phenyl, substituted phenyl, naphthyl, cycloalkyl, propargyl orallyl; and

R⁴ is aryl, heteroaryl or cycloalkyl. In particular embodiments, R⁴ is2-furyl, 2-thiophene or 3-pyridyl.

Methods of using the kappa opioid receptor antagonist or agonist toselectively modulate the activity of kappa opioid receptor in vitro andin the treatment of a patient with, e.g., an ethanol use disorder, ananxiety disorder, a depressive illness, schizophrenia, mania orpost-traumatic stress disorder are also provided.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows plasma and brain concentrations of two representativetriazole analogues (KUIP10051N, FIG. 1A; and KUC1404186N, FIG. 1B)administered to 2 month old mice (10 mg/kg, i.p.). Plasma and brainswere collected at and 60 minutes from two groups of mice (n=3).Concentrations were determined from standard curves prepared in theappropriate matrix. Presented are the calculated means±SEM.

DETAILED DESCRIPTION OF THE INVENTION

Using a high throughput screening (HTS) campaign and subsequent hitoptimization, new effector (antagonist and agonist) chemotypes of theKOP receptor (KOR) have now been developed. These distinct classes ofKOR ligands were developed based on initial hit compounds and subsequentoptimization through the synthesis of additional analogues toinvestigate the Structure-Activity Relationship (SAR).

KOR Antagonists.

When compared with known KOR antagonists such as 5′-guanidinonaltrindoleand JDTic, the instant pyridopyrrolo pyrazinone antagonist (Chemotype I,Formula I) is unique with a simplified, modular structure.

This chemotype of KOR antagonists is of use in selectively inhibitingthe human kappa opioid receptor to provide a scientific tool useful inhelping to elucidate individual brain pathways that underlie addictivebehavior, thus enabling improved understanding of the molecular basis ofdependency and potentially providing a basis for therapeuticdevelopment. Moreover, given that the instant compounds are in the rangeof potencies for administration to animals, these antagonists, as wellas analogs and derivatives therefore find use in the treatment ofaddiction, prevention of reinstatement of drug taking behavior, blockingaspects of nicotine withdrawal, and treatment of depression andposttraumatic stress disorder (PTSD).

Accordingly, the present invention provides kappa opioid antagoniststhat bind to kappa opioid receptors with high affinity and/orspecificity. Antagonists of the present invention are those representedby the Formula I:

wherein

X, Y and Z are independently selected from H,H; O; S or NH;

R¹ is H, a halogen group (e.g., F, I, Cl or Br), or a substituted orunsubstituted lower alkyl or alkoxy group; and

R² is present or absent, and when present is a substituent on one ormore ring atoms (e.g., position 2, 3, or 4) and is for each ring atomindependently H, a halogen group, or a substituted or unsubstitutedlower alkyl or alkoxy group.

KOR Agonists.

The structure of the instant agonists of Chemotype II (Formula II) aredistinct from known KOR agonists such as Salvinorin A, Ketazocine andU-50488.

Evidence indicates that activation of KOR opposes a variety ofMOR-mediated actions throughout the brain and spinal cord (Pan (1998)Trends Pharmacol. Sci. 19(3):94-8). Studies indicate that antagonism ofendogenous KOR apparently elicits a potentiating effect on somemorphine-withdrawal signs, including weight loss. Stimulation ofendogenous KOR is therefore of use in attenuating morphine withdrawalsymptoms. In this respect, Dynorphin A, a know KOR agonist, has beenreported to inhibit morphine withdrawal symptoms induced by naloxoneprecipitation or morphine discontinuation in morphine-dependent animals(Suzuki, et al. (1992) Life Sci., 50(12):849-56). Therefore, not only isChemotype II of use as a scientific tool to elucidate individual brainpathways that underlie addictive behavior, these agonists are useful inthe treatment of withdrawal. Moreover, the instant agonists, as well asanalogs, derivatives and partial agonists therefore, find use in thetreatment of the manic phase of bipolar disorder, among otherconditions.

Accordingly, the present invention provides kappa opioid agonists thatbind to kappa opioid receptors with high affinity and/or specificity.Agonist compounds of the present invention are those represented by theFormula II:

wherein

X′ is hydrogen or Br;

each Z′ is independently C, CH or N;

n is 0 or 1;

R³ is phenyl, substituted phenyl, naphthyl, cycloalkyl, propargyl orallyl; and

R⁴ is aryl, heteroaryl or cycloalkyl. In particular embodiments, R⁴ is2-furyl, 2-thiophene or 3-pyridyl.

For the compounds of this invention, the term “lower alkyl” is intendedto mean a branched or unbranched saturated monovalent hydrocarbonradical containing 1 to 6 carbon atoms, such as methyl, ethyl, propyl,isopropyl, tert-butyl, butyl, n-hexyl and the like. Similarly, a loweralkoxy group is a C1-C6 alkoxy group, such as methoxy, ethoxy, oracetoxy.

“Cycloalkyl” means a monocyclic or fused bicyclic, saturated orpartially unsaturated, monovalent hydrocarbon radical of three to tencarbon ring atoms. Unless otherwise stated, the valency of the group maybe located on any atom of any ring within the radical, valency rulespermitting. More specifically, the term cycloalkyl includes, but is notlimited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl,decahydronaphthyl (including, but not limited to decahydronaphth-1-yl,decahydronaphth-2-yl, and the like), norbornyl, adamantly, orcyclohexenyl, and the like. The cycloalkyl ring may be unsubstituted orsubstituted with one or more substituents which may be the same ordifferent, and are as defined herein.

The term “aryl” refers to single-ring aromatic radicals which mayinclude from 5 to 20 carbon atoms. Aryl groups include, but are notlimited to, phenyl, biphenyl, anthracenyl, and naphthenyl. The phrase“substituted aryl group” refers to an aryl group that is substitutedwith one or more substituents.

The phrase “heteroaryl” refers to a 3 to 20-membered aromatic ringcomposed of carbon atoms and heteroatoms, such as N, S, and O. Theheteroaryl ring may be attached at any heteroatom or carbon atom.Representative heteroaryl compounds include, for example, imidazolyl,pyridyl, pyrazinyl, pyrimidinyl, thiophenyl, thiazolyl, furanyl,pyridofuranyl, pyrimidofuranyl, pyridothienyl, pyridazothienyl,pyridooxazolyl, pyridazooxazolyl, pyrimidooxazolyl, pyridothiazolyl,pyridazothiazolyl, pyrrolyl, pyrrolinyl, imidazolyl, pyrazolyl, pyridyl,dihydropyridyl, pyrimidyl, pyrazinyl, pyridazinyl, triazolyl (e.g.,4H-1,2,4-triazolyl, 1H-1,2,3-triazolyl, and 2H-1,2,3-triazolyl),tetrazolyl, (e.g., 1H-tetrazolyl and 2H tetrazolyl), pyrrolidinyl,imidazolidinyl, piperidinyl, piperazinyl, indolyl, isoindolyl,indolinyl, indolizinyl, benzimidazolyl, quinolyl, isoquinolyl,indazolyl, benzotriazolyl, oxazolyl, isoxazolyl, oxadiazolyl (e.g.,1,2,4-oxadiazolyl, 1,3,4-oxadiazolyl, and 1,2,5-oxadiazolyl),benzoxazolyl, benzoxadiazolyl, benzoxazinyl (e.g., 2H-1,4-benzoxazinyl),thiazolyl, isothiazolyl, thiadiazolyl (e.g., 1,2,3-thiadiazolyl,1,2,4-thiadiazolyl, 1,3,4-thiadiazolyl, and 1,2,5-thiadiazolyl). Thephrase “substituted heteroaryl” refers to a heteroaryl group that issubstituted with one or more substituents.

Exemplary substituent groups of lower alkyl, alkoxy, cycloalkyl, aryl,phenyl and heteroaryl groups include, but are not limited to, one ormore halogen groups (i.e., fluorine, chlorine, bromine and iodine),lower alkyl groups, lower alkoxy groups, alkenyl groups (e.g., C2-C6),hydroxyl groups, amine groups, amide groups, nitro groups, nitrosogroups, aldehyde groups, carboxyl groups, sulhydryl groups, ═O, —CF₃,—CN, and carbonothioyl groups.

The antagonist and agonist compounds of the present invention, includinganalogs, derivatives and partial agonists thereof, are selective for thekappa receptor. By “selective kappa antagonist” is meant any chemicalcompound which has affinity for the kappa opioid receptor, substantiallyno agonist activity, and produces less than 15% of the maximal responsein comparison to dynorphin A. The selective kappa antagonist has morethan 5, 10, 25, 50, 100, 200, 300, 500, 700, 1,000, or 2,000 foldgreater affinity for kappa opioid receptors than for each of the mu anddelta opioid receptors. Affinities for the various opioid receptorsubtypes are determined using standard in vitro assays. For example, thebinding assays may be conducted as described herein or may utilizeguinea pig brain membranes or stably transfected Chinese Hamster Ovary(CHO) cells expressing each of the three opioid receptors. In particularembodiments, the instant compounds are specific for the kappa opioidreceptor and exhibit greater than 100-fold affinity over the mu opioidreceptor and 10-fold affinity over the delta opioid receptor.

By “selective kappa receptor partial agonist” is meant a chemicalcompound which has affinity for the kappa opioid receptor and exhibitsagonist activity, but produces only a partial (i.e., submaximal)response of between 15% and 85% in comparison to dynorphin A, anendogenous neurotransmitter of the kappa opioid receptor. The selectivekappa partial agonist has more than 5, 10, 25, 50, 100, 200, 300, 500,700, 1,000, or 2,000 fold greater affinity for kappa opioid receptorsthan for each of the mu and delta opioid receptors.

By “selective kappa receptor agonist” is meant a chemical compound whichhas affinity for the kappa opioid receptor, exhibits agonist activity,and produces at least 85% of the maximal response in comparison todynorphin A. The selective kappa agonist has more than 5, 10, 25, 50,100, 200, 300, 500, 700, 1,000, or 2,000 fold greater affinity for kappaopioid receptors than for each of the mu and delta opioid receptors.

A most preferred set of antagonist compounds of Formula I includes thecompounds as shown in Table 1. A most preferred set of agonist compoundsof Formula II includes the compounds as shown in Table 8. Such compoundscan be prepared as pharmaceutical compositions, pharmaceuticallyacceptable derivatives, or pharmaceutically acceptable salts and beprovided alone or in combination in the form of a kit with unit doses ofthe subject compounds. In such kits, in addition to the containerscontaining the unit doses will be an informational package insertdescribing the use and attendant benefits of using the compound(s).

A “pharmaceutically acceptable derivative” of a compound of theinvention include salts, esters, enol ethers, enol esters, acetals,ketals, orthoesters, hemiacetals, hemiketals, acids, bases, solvates,hydrates or prodrugs thereof. Such derivatives may be readily preparedby those of skill in this art using known methods for suchderivatization. The compounds produced may be administered to animals orhumans without substantial toxic effects and either are pharmaceuticallyactive or are prodrugs.

A “pharmaceutically acceptable salt” of a compound means a salt that ispharmaceutically acceptable and that possesses the desiredpharmacological activity of the parent compound. Such salts include: (1)acid addition salts, formed with inorganic acids such as hydrochloricacid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, andthe like; or formed with organic acids such as acetic acid, propionicacid, hexanoic acid, cyclopentanepropionic acid, glycolic acid, pyruvicacid, lactic acid, malonic acid, succinic acid, malic acid, maleic acid,fumaric acid, tartaric acid, citric acid, benzoic acid,3-(4-hydroxybenzoyl)benzoic acid, cinnamic acid, mandelic acid,methanesulfonic acid, ethanesulfonic acid, 1,2-ethanedisulfonic acid,2-hydroxyethanesulfonic acid, benzenesulfonic acid,4-chlorobenzenesulfonic acid, 2-naphthalenesulfonic acid,4-toluenesulfonic acid, camphorsulfonic acid, glucoheptonic acid,4,4′-methylenebis-(3-hydroxy-2-ene-1-carboxylic acid), 3-phenylpropionicacid, trimethylacetic acid, tertiary butylacetic acid, lauryl sulfuricacid, gluconic acid, glutamic acid, hydroxynaphthoic acid, salicylicacid, stearic acid, muconic acid, and the like; or (2) salts formed whenan acidic proton present in the parent compound either is replaced by ametal ion, e.g., an alkali metal ion, an alkaline earth ion, or analuminum ion; or coordinates with an organic base such as ethanolamine,diethanolamine, triethanolamine, tromethamine, N-methylglucamine, andthe like.

In addition to the compounds specifically disclosed herein, the presentinvention also pertains to analogs, derivatives and partial agonists ofsaid compounds that modulate (i.e., agonize or antagonize) KOR activity.Broadly, the KOR three-dimensional crystal structure in combination withthe structure of the instant compounds can be used to design or screenfor a test compound with KOR modulatory activity; and the compounddesigned or screened for can be tested for its ability to selectivelyagonize or antagonize the activity of KOR.

The receptor selectivities discussed above are determined based on thebinding affinities at the receptors indicated or their selectivity inopioid functional assays exemplified herein including, but not limitedto a [³⁵S]GTPγ-S assay, the DISCOVERX PATHHUNTER β-arrestin recruitmentassay and/or a high content imaging β-arrestin translocation assay.

The compounds of the present invention can be used to bind kappa opioidreceptors. Such binding can be accomplished by contacting the receptorwith an effective amount of the inventive compound. Of course, suchcontacting is preferably conducted in an aqueous medium, preferably atphysiologically relevant ionic strength, pH, etc.

The inventive compounds can also be used to treat patients havingdisease states which are ameliorated by binding opioid receptors or inany treatment wherein temporary suppression or activation of the kappaopioid receptor system is desired. Specifically, kappa opioid receptorantagonists are of use in the treatment of opiate addiction (such asheroin addiction), or cocaine addiction as well as being useful ascytostatic agents, as anti-migraine agents, as immunomodulators, asimmunosuppressives, as antiarthritic agents, as anti-allergic agents, asvirucides, to treat diarrhea, as antipsychotics, as anti-schizophrenics,as anti-depressants, as uropathic agents, as antitussives, asanti-addictive agents, as anti-smoking agents, to treat alcoholism, ashypotensive agents, to treat and/or prevent paralysis resulting fromtraumatic ischemia, general neuroprotection against ischemic trauma, asadjuncts to nerve growth factor treatment of hyperalgesia and nervegrafts, as anti-diuretics, as stimulants, as anti-convulsants, or totreat obesity. Additionally, the present antagonists can be used in thetreatment of Parkinson's disease as an adjunct to L-dopa for treatmentof dyskinesia associated with the L-dopa treatment. They can also beused with kappa agonists disclosed herein or those known in the art(e.g., U-50,488, Salvinorin A, and Ketazocine) as analgesics, or for anycondition requiring suppression of the kappa receptor system. Selectivekappa receptor agonists of the invention are particularly useful fortreating mania associated with bipolar disorder, acute mania, andchronic mania.

Post-traumatic Stress Disorder (PTSD) is an anxiety disorder that candevelop after exposure to a traumatic event. Emerging evidence suggeststhat opiate systems may modulate the development and expression of PTSD.Mu opioid receptor (MOR) analgesics, such as morphine, are often givenas a response to trauma, and there is emerging evidence that they are,at least partially, protective against PTSD. The kappa opioid receptor(KOR) system has also been implicated in stress-related processes, withKOR agonists reported to enhance stress in both laboratory animals andin humans, and KOR antagonists reported to attenuate stress-likebehaviors. Therefore, the KOR antagonists of this invention also finduse in the treatment of trauma and in reducing the emergence andpersistence of PTSD.

As used herein, the term “subject” or “patient” is intended to includeliving organisms in which certain conditions as described herein canoccur. Examples include humans, monkeys, cows, sheep, goats, dogs, cats,mice, rats, and transgenic species thereof. In a particular embodiment,the subject is a primate. In a specific embodiment, the primate is ahuman. Other examples of subjects include experimental animals such asmice, rats, dogs, cats, goats, sheep, pigs, and cows. The experimentalanimal can be an animal model for a disorder, e.g., a transgenic mousewith an Alzheimer's-type neuropathology.

The compounds of the present invention can be administered in aneffective amount by any of the conventional techniques well-establishedin the medical field. For example, the compounds can be administeredorally, intraveneously, or intramuscularly. When so administered, theinventive compounds can be combined with any of the well-knownpharmaceutical carriers and additives that are customarily used in suchpharmaceutical compositions. For a discussion of dosing forms, carriers,additives, pharmacodynamics, etc., see Kirk-Othmer Encyclopedia ofChemical Technology, Fourth Edition, Vol. 18, 1996, pp. 480-590. Thepatient is preferably a mammal, with human patients especiallypreferred. Effective amounts are readily determined by those of ordinaryskill in the art.

Active compounds are administered at a therapeutically effective dosagesufficient to treat a subject. An “effective amount” desirably reducesthe amount of symptoms of the condition in the subject by at least about20%, more preferably by at least about 40%, even more preferably by atleast about 60%, and still more preferably by at least about 80%relative to untreated subjects. For example, the efficacy of a compoundcan be evaluated in an animal model system that is predictive ofefficacy in treating the disease in humans.

Compounds of this invention can be administered as a single dosage perday, or as multiple dosages per day. When administered as multipledosages, the dosages can be equal doses or doses of varying amount,based upon the time between the doses (i.e., when there will be a longertime between doses, such as overnight while sleeping, the doseadministered will be higher to allow the compound to be present in thebloodstream of the patient for the longer period of time at effectivelevels). Preferably, the compound and compositions containing thecompound are administered as a single dose or from 2-4 equal doses perday.

Pharmaceutical compositions containing the present compounds typicallyinclude a physiologically acceptable carrier, such as buffer orconventional pharmaceutical solid carriers, and if desired, one or moreother excipients.

The invention is described in greater detail by the followingnon-limiting examples.

Example 1 Screening and Hit Identification

The distinct class of KOR ligands presented herein was developed basedon initial hit compounds uncovered through the screening of theMolecular Libraries Small Molecule Repository (MLSMR) compoundcollection and subsequent optimization through the synthesis ofadditional analogues to investigate SAR. The project was initiated todevelop new chemical probes and it was contemplated that the interest inelucidating the pharmacology of the KOR would benefit from a diversecollection of readily-available ligands. Ideally, these ligands wouldpossess a range of pharmacological profiles. For a compound to beinteresting as a lead for a pharmacological probe, it must be selectivefor the KOR over the other opioid receptors and possess sufficientpotency to be useful. It was determined that a 100-fold selectivity and1 μM potency was the minimum criteria for an interesting lead compoundof each chemotype. Moreover, any new chemotype could not resemble anyalready known opioid ligand. Based on these requirements, MLPCN probeantagonist compounds were analyzed (Hedrick, et al. Selective KOPreceptor agonists, PMID: 21433386, PUBCHEM AID 1786; Hedrick, et al.Selective KOP receptor antagonists, PMID: 21433381, PUBCHEM AID 1785).

In this campaign, two assay platforms were employed to evaluate KORactivity and selectivity: the KOR1 DISCOVERX β-Arrestin PATHHUNTER assayand an imaging based β-arrestin translocation assay for confirmatory andselectivity assays. The KOR1 DISCOVERX β-Arrestin PATHHUNTER assay is acommercial platform for direct measurement of GPCR (G protein-coupledreceptor) activation by detection of β-Arrestin binding to the KOR. Inthis system, β-Arrestin is fused to an N-terminal deletion mutant ofβ-galactosidase (termed the enzyme acceptor or EA) and the GPCR ofinterest is fused to a smaller (42 amino acid), weakly complementingfragment (termed PROLINK). In cells that stably express these fusionproteins, ligand stimulation results in the recruitment of β-Arrestin bythe GPCR. This interaction encourages the complementation of the twoβ-galactosidase fragments resulting in the formation of a functionalenzyme that converts substrate in the assay medium to a detectablesignal. The imaging based high-content β-arrestin translocation assay isbased upon the redistribution of β-Arrestin linked to green fluorescentprotein (GFP) to the cell surface and detection (Barak, et al. (1997) J.Biol. Chem. 272:27497-27500).

The minimum criteria for a successful probe compound were set as an IC₅₀of less than 1 μM in the KOR1 DISCOVERX β-Arrestin assay and greaterthan 100-fold selective for the KOR over either the MOR (mu opioidreceptor) or DOR (delta opioid receptor) in the β-arrestin translocationcomparison assay (or within the detection limits of the assay platform).Moreover, it was of importance to identify KOR agonists and antagonistsdifferent from probes known in the art, as well as scaffolds that wouldbe chemically attractive in terms of synthetic accessibility andstructural malleability.

Initially, a portion (approximately 290,000 compounds) of the MLSMRcompound collection was tested in the KOR1 DISCOVERX β-arrestin primarykappa opioid (KOP) agonist and antagonist screens (PUBCHEM AID 1777 and1778, respectively) at a single concentration point (10 μM). Compoundswith an activity >50% were retested with compound solutions resuppliedfrom the MLSMR collection to confirm single concentration activity. Theconfirmed compounds were further tested in concentration responsescreens using the DISCOVERX β-arrestin primary KOP agonist andantagonist screen to obtain EC₅₀ or IC₅₀ values (PUBCHEM AID 2284 and2285, respectively). These compounds were concurrently tested in aβ-galactosidase counterscreen assay (PUBCHEM AID 1966) to assess thepossibility that these compounds might inhibit the reporting enzyme. Theactivity of the validated compounds was confirmed in the KOR agonist andantagonist β-arrestin translocation assay (PUBCHEM AID 2359 and 2348,respectively). The KOR:DOR:MOR selectivity of the compounds wasdetermined using the analogous β-arrestin translocation assays for theDOR (PUBCHEM AID 2370 and 2357, for agonists and antagonist,respectively) and the MOR (PUBCHEM AID 2352 and 2420, for agonists andantagonists, respectively). The minimum threshold for the identificationof an interesting new chemotype was set as an IC₅₀ (agonist) or EC₅₀(antagonist) of less than 1 μM in the KOR PATHHUNTER assay and greaterthan 100-fold selective for the KOR over either the MOR or DOR in theβ-arrestin translocation assay (or within the detection limits of theassay platform). Following data analysis and attrition of compounds fromthe counterscreen, novel and selective classes of compounds emerged fromthe screens possessing a strong potential for optimization. Thechemotype (Chemotype I) of antagonistic compounds was based upon therepresentative achiral compound,N—N-(3-(4-(4-methoxyphenyl)piperazin-1-yl)propyl)-2-(1-methyl-6-oxopyrido[2,3-e]pyrrolo[1,2-a]pyrazin-5(6H)-yl)acetamide(PUBCHEM Compound ID 44665680, Substance ID 88442997), referred toherein as compound 1{3}.

The chemotype (Chemotype II) of agonistic compounds was based upon thefollowing representative compound, referred to herein as compound 2{6}(PUBCHEM Compound ID 2482316, Substance ID 17388459).

Example 2 Synthesis of Compound 1{3}

Compound 1{3} was readily synthesized by the synthetic route shown inScheme 1. Of particular note, compound 1{3} is achiral, which is incontrast to conventional KOR antagonists.

2-Fluoro-4-methyl-3-(1H-pyrrol-1-yl)pyridine

2-Fluoro-4-methylpyridin-3-amine (1.0 g, 7.93 mmol) and2,5-dimethoxytetrahydrofuran (1.08 mL, 1.05 equiv.) were suspended in 3mL of acetic acid and refluxed for 2 hours. The reaction was cooled downto room temperature. The solvents were removed and the residue waspurified by silica gel chromatography (EtOAc/hexanes=1:8, Rf=0.3) toafford 1.0 g (72%) oil. ¹H NMR (400 MHz, CDCl₃) δ 8.10 (dd, J=0.8, 5.1Hz, 1H), 7.17 (d, J=5.1 Hz, 1H), 6.74 (td, J=2.1, 0.9 Hz, 2H), 6.40 (t,J=2.1 Hz, 2H), 2.26 (s, 3H). ¹³C NMR (101 MHz, CDCl₃) δ 160.2, 157.8,149.6, 149.5, 145.5, 145.4, 123.80, 123.76, 122.1, 109.8, 17.29, 17.25.HRMS (m/z) calcd for C₁₀H₁₀FN₂ (M+H) 177.0828; found 177.0827. Richards,et al. (2008) Bioorg. & Med. Chem. Lett. 18:4325-27.

4-Methyl-3-(1H-pyrrol-1-yl)pyridine-2-amine

2-Fluoro-4-methyl-3-(1H-pyrrol-1-yl)pyridine (3.9 g, 22.1 mmol) wasdissolved in 80 mL of ammonia solution (7N in MeOH) in a sealed tube(350 mL). The mixture was heated at 150° C. for 2 days protected with ablast shield. The mixture was cooled to room temperature, then cooled inthe ice for minutes. The filtrate was evaporated to dryness and purifiedby flash chromatography (EtOAc/Hexanes=1:1, Rf=0.5) to give 2.9 g (76%)white solid. ¹H NMR (400 MHz, CDCl₃) δ 7.96 (d, J=5.1 Hz, 1H) 6.67 (t,J=2.1 Hz, 2H), 6.60 (d, J=5.2 Hz, 1H), 6.41 (t, J=2.1 Hz, 2H), 4.52 (s,2H), 2.02 (s, 3H). ¹³C NMR (101 MHz, CDCl₃) δ 156.1, 147.2, 145.9,121.5, 121.1, 116.0, 110.0, 16.7. HRMS (m/z): calcd for C₁₀H₁₂N₃ (M+H)174.1031; found 174.1026. Peet & Sunder (1986) Heterocycles24:3213-3221.

1-Methylpyrido[2,3-e]pyrrolo[1,2-a]pyrazin-6(5H)-one

4-Methyl-3-(1H-pyrrol-1-yl)pyridine-2-amine (1.0 g, 5.8 mmol) andtriphosgene (2.6 g, 8.7 mmol) were dissolved in 100 mL of toluene. Themixture was refluxed for 3 hours, then cooled to room temperature. Thered solid was collected after filtration and washed with CH₃CN 0.5 g(43%). The material was used directly for the next step reaction withoutpurification. ¹H NMR (400 MHz, DMSO) δ 11.64 (s, 1H), 8.15 (dd, J=1.4,2.9 Hz, 1H), 8.13 (d, J=4.9 Hz, 1H), 7.20-7.08 (m, 2H), 6.75 (dd, J=2.9,3.9 Hz, 1H), 2.81 (s, 3H).

Methyl2-(1-methyl-6-oxopyrido[2,3-e]pyrrolo[1,2-a]pyrazin-5(6H)-yl)acetate

To a solution of 1-methylpyrido[2,3-e]pyrrolo[1,2-a]pyrazin-6(5H)-one(50 mg, 0.25 mmol) in 2 mL of DMF, was added NaH (60%, 11 mg, 0.28mmol). The mixture was stirred at room temperature for 1 hour.Methylbromoacetate (26 mL, 0.28 mmol) was added. The mixture was stirredfor 16 hours. Solvents were removed under vacuum and the residue waspurified by silica gel flash chromatography. (DCM/MeOH=1:10, Rf=0.5) toafford 37 mg (54%) light yellow solid. ¹H NMR (400 MHz, CDCl₃) δ 8.15(d, J=4.9 Hz, 1H), 7.95 (dd, J=1.5, 2.9 Hz, 1H) 7.36 (dd, J=1.5, 4.0 Hz,1H), 7.00 (d, J=4.9 Hz, 1H), 6.71 (dd, J=2.9, 4.0 Hz, 1H), 5.25 (s, 2H),3.77 (s, 3H), 2.83 (s, 3H). ¹³C NMR (101 MHz, CDCl₃) δ 169.3, 155.5,143.1, 142.1, 134.8, 124.2, 122.7, 122.6, 120.4, 113.5, 113.4, 52.3,41.9, 22.9. HRMS (m/z): calcd for C₁₄H₃₄N₃O₃ (M+H) 272.1035; found272.1042.

2-(1-Methyl-6-oxopyrido[2,3-e]pyrrolo[1,2-a]pyrazin-5(6H)-yl)acetic acid

Methyl2-(1-methyl-6-oxopyrido[2,3-e]pyrrolo[1,2-a]pyrazin-5(6H)-yl)acetate(517 mg, 1.91 mmol) was dissolved in 20 mL of MeOH/H₂O/THF (1:1:4). LiOH(68.5 mg, 2.86 mmol) was added. The mixture was stirred at roomtemperature for 16 hours. The solvents were removed and residue wasdissolved in water, washed with ether, then neutralized with 2N HCl topH=3. 356 mg (73%) white solid was obtained after filtration and driedunder vacuum. ¹H NMR (400 MHz, DMSO) δ 12.90 (s, 1H), 8.27-8.17 (m, 2H),7.29-7.19 (m, 2H), 6.80 (dd, J=2.9, 3.9 Hz, 1H), 5.02 (s, 2H), 2.85 (s,3H). ¹³C NMR (101 MHz, DMSO, APT) δ 169.8, 154.5, 143.1, 141.4, 135.9,123.9, 123.3, 122.8, 119.5, 113.3, 112.7, 41.6, 22.2. HRMS (m/z): calcdfor C₁₃H₁₂N₃O₃ (M+H) 258.0879; found 258.0894.

3-(4-(4-Methoxyphenyl)piperazin-1-yl)propanenitrile

4-Methoxyphenypiperazine (0.92 g, 4.68 mmol) and acrylonitrile (0.31 mL,4.68 mmol) were mixed in a 10 mL reaction tube and stirred for 16 hours.The product was purified by silica gel flash chromatography(EtOAc/hexanes=1:8, Rf=0.3) to give 0.8 g (74%) white solid. ¹H NMR (400MHz, CDCl₃) δ 6.98-6.90 (m, 2H), 6.90-6.82 (m, 2H), 3.79 (s, 3H),3.17-3.07 (m, 4H), 2.78 (t, J=7.0 Hz, 2H), 2.74-2.64 (m, 4H), 2.57 (t,J=7.0 Hz, 2H). ¹³C NMR (101 MHz, CDCl₃) δ 154.0, 145.5, 118.8, 118.4,114.5, 55.6, 53.4, 52.8, 50.6, 15.9. Upadhayaya, et al. (2004) Bioorg.Med. Chem. 12:2225-2238.

3-(4-(4-Methoxyphenyl)piperazin-1-yl)propan-1-amine

A solution of 3-(4-(4-methoxyphenyl)piperazin-1-yl)propanenitrile (0.8g, 3.26 mmol) in 15 mL ether was added to the suspension of LiAlH₄ (0.19g, 4.89 mmol) in 5 mL of ether. The mixture was stirred at roomtemperature for 16 hours, then quenched with 2N NaOH (1 mL). The etherphase was dried over MgSO₄ and evaporated to dryness to give 0.68 g(84%) white solid, which was used directly without further purification.¹H NMR (400 MHz, CDCl₃) δ 6.89 (d, J=9.1 Hz, 2H), 6.82 (d, J=9.1 Hz,2H), 3.75 (s, 3H), 3.15-3.04 (m, 4H), 2.87 (s, br. 2H), 2.76 (t, J=6.8Hz, 2H), 2.67-2.53 (m, 4H), 2.51-2.37 (m, 2H), 1.75-1.54 (m, 2H). ¹³CNMR (101 MHz, CDCl₃) δ 153.8, 145.7, 118.1, 114.4, 56.4, 55.5, 53.48,50.6, 40.6, 30.1. Valenta, et al. (1990) Collect. Czech. Chem. Commun.55:797-808.

N-(3-(4-(4-Methoxyphenyl)piperazin-1-yl)propyl)-2-(1-methyl-6-oxopyrido[2,3-e]pyrrolo[1,2-a]pyrazin-5(6H)-yl)acetamide

2-(1-Methyl-6-oxopyrido[2,3-e]pyrrolo[1,2-a]pyrazin-5(6H)-yl)acetic acid(30 mg, 0.12 mmol), 3-(4-(4-methoxyphenyl)piperazin-1-yl)propan-1-amine(43.6 mg, 0.17 mmol) and DMAP (1.4 mg, 0.012 mmol) were dissolved in 1mL of DCM. Diisopropylcarbodiimide (0.09 mL, 0.58 mmol) was added. Themixture was stirred at room temperature for 16 hours and the product waspurified by silica gel flash chromatography (DCM/MeOH=10:1, Rf=0.5) togive 30 mg (53%) white solid. ¹H NMR (400 MHz, CDCl₃) δ 8.17 (d, J=4.9Hz, 1H), 7.87 (dd, J=1.4, 2.9 Hz, 1H), 7.33 (dd, J=1.4, 4.0 Hz, 1H),7.12 (s, 1H), 6.99 (d, J=5.0 Hz, 1H), 6.84 (s, 4H), 6.68 (dd, J=2.9, 4.0Hz, 1H), 5.11 (s, 2H), 3.78 (s, 3H), 3.41 (dd, J=5.8, 12.0 Hz, 2H),3.02-2.91 (m, 4H), 2.74 (s, 3H), 2.63-2.54 (m, 4H), 2.49 (t, J=6.4 Hz,2H), 1.75-1.69 (m, 2H). ¹³C NMR (101 MHz, CDCl₃) δ 167.9, 155.8, 153.8,145.4, 143.2, 142.3, 134.8, 124.1, 122.8, 122.6, 120.4, 118.0, 114.4,113.5, 113.4, 57.3, 55.6, 53.4, 50.4, 44.2, 39.4, 25.2, 22.8. HRMS(m/z): calcd for C₂₇H₃₃N₆O₃ (M+H) 489.2609; found 489.2600.

Example 3 SAR Expansion and Optimization of Chemotype I

Synthetic chemistry was conducted for the new chemotype, and theanalogues synthesized effectively represent a single round ofoptimization, therefore the decision of analogues synthesized was guidedby the initial screening data and a limited set ofcommercially-available follow up compounds. Therefore, furtheroptimization of the chemotype is contemplated. The short, flexiblesynthetic route of the instant scaffold allows easy preparation ofadditional analogs to hone in on the desired characteristics or allowthe possible incorporation of mass detection or fluorescent markers.Both the malleability and optimization potential is illustrated throughthe following detailed account for the scaffold. A key feature of theinstant chemotype is that it is a distinct, novel class of general KORantagonists with sufficient analogues to demonstrate tractable SAR. Themodular synthetic route to this class of compounds is summarized inScheme 2.

Example 4 Chemotype I Analogues

The HTS campaign for antagonists afforded the Chemotype I compounds withpromising potency (compounds 1{1} and 1{2}, Table 1) as well as a numberof analogues found to have IC₅₀ values above ten micromolar (Table 2).Notably, both compounds 1{1} and 1{2} contained a p-methoxyphenylsubstituted piperazine moiety and a pyrrolopyrazinone core scaffold. Anamide coupling-based synthetic route was developed to synthesize thischemotype, allowing the synthesis of 18 analogues in a single round ofSAR. The synthetic efforts were augmented with the purchase of eightcommercial analogues and a selection of the screening results ispresented in Tables 1 and 2.

TABLE 1

Entry 1 {n} CID SID A R¹ R²  1 22553442 87544476 N H 4-OMe 8721879090340552  2 22522554 87218788 C H 4-OMe  3 44665680 88442997 N Me 4-OMe 4 44665679 88443000 N Me 2,4-diOMe  5 44665685 88442999 N Me 3,4-OCH₂O 6 44665687 88442998 N Me 4-Cl  7 44665686 88442996 N Me 4-Me  844665682 88442991 N Me H  9 44828478 90340555 N H 2,4-diOMe 10 4482847990340554 N H 3,4-OCH₂O 11 44828476 90340553 N H 4-Cl 12 4482848090340551 N H 4-Me 13 44828477 90340550 N H H

Entry 1 {n} CID SID A R¹ R² 14 45100475 92093149 N Br 4-OMe 15 4510047492093150 N Br 4-Cl 16 45100477 92093151 N Br 2,4-diOMe 17 4510047692093152 N Br 3,4-OCH₂O CID, PUBCHEM Chemical ID; SID, PUBCHEM SubstanceID.

TABLE 2 Potency (μM) Average ± S.E.M. (stdv/sqrt (n)) (n = replicates)Target Antitarget KOR MOR DOR Entry KOR HCS HCS HCS 1{n} n DRX (n = 2)(n = 2) (n = 2) 1 12 1.97  1.31 (n = 8) >32 >32 2 4.32 15.3 >32 >32 3 80.12 0.003 (n = 4) >32 >32 4 8 0.19 0.004 (n = 6) 30.9 >32 5 8 0.430.045 (n = 4) 17.2 >32 6 8 0.69 0.07 >32 >32 7 4 0.88 2.28 >32 >32 8 44.95 2.56 22.5 >32 9 4 2.65 0.49 10.8 >32 10 4 4.23 0.78 >32 >32 11 85.18 0.096 >32 >32 12 4 3.68  0.89 (n = 4) 18.8 (n = 4) >32 13 2 >20 12.7 (n = 4) >32 >32 14 4 3.05 0.69 10.3 >32 15 4 >20 >32 5.32 >32 16 42.82 3.02 10.22 >32 17 8 3.11 0.15 >32 >32 n value in column refers toKOR DRX replicates.

Unexpectedly, the incorporation of a single methyl group on theheterocyclic core increased the potency of the p-methoxyphenylsubstituted piperazine analogue by over ten fold in the β-arrestin assayand afforded the KOR-selective compound 1{3} with a high contentβ-arrestin translocation assay IC₅₀ of 3 nM. While less potent thanJDTic (IC₅₀=0.02 nM), this compound was over 10,000-fold selective forthe KOR over both the DOR and MOR, an improved selectivity compared toJDTic (202-fold selective for the KOR over the MOR). Based on thesemerits, compound 1{3} was nominated as an MLPCN probe compound. Severalother compounds sharing the additional methyl group also exceeded theprobe criteria to a lesser extent (Table 2, compound 1{4} through 1{7}).For several compounds, a slight loss in selectivity against the MORcounterscreen was observed (compounds 1{4}, 1{5}, 1{8}, 1{9}, 1{12},1{14} and 1{16}). This trend was amplified for compound 1{16}, which wasa MOR-selective antagonist of modest potency (5.32 μM). The presentprobe candidate could be utilized for additional studies in this area orfurther refined through additional rounds of SAR to optimize thisdisparity, further increase potency or improve other desirablecharacteristics. This scaffold is highly amenable to modification viathe present synthetic route and various substituents of this chemotypeare contemplated.

Moreover SAR analysis of the phenylpiperazine moiety was conducted(Table 3). It was observed that these analogues conferred a drastic lossin activity (Table 4). The compounds in entries 1{26} (CID 45479166) and1{27} (CID 45479168) demonstrated the connection between tether lengthand activity, wherein the lower activity of both the two carbon and fourcarbon tether analogues indicates that the three-carbon tether length isoptimal. It was noted that entries 1{24} (CID 22553452) and 1{25} (CID22553453) could provide lead structures for a MOP receptor-selectivecompound based on the present scaffold.

TABLE 3

Entry 1 {n} CID SID R¹ R³ 19 22553408 87544473 H

20 22553432 87544474 H

21 22553433 87544475 H

22 22553447 87544477 H

23 22553448 87544478 H

24 22553452 87544479 H

25 22553453 87544480 H

26 45479166 93575685 Me

27 45479168 93575686 Me

CID, PUBCHEM Chemical ID; SID, PUBCHEM Substance ID.

TABLE 4 Potency (μM) Average ± S.E.M. (stdv/sqrt (n)) (n = replicates)Target Antitarget KOR MOR DOR Entry KOR HCS HCS HCS 1{n} n DRX (n = 2)(n = 2) (n = 2) 19 4 >20 >32 >32 >32 20 4 >20 >32 >32 >32 214 >20 >32 >32 >32 22 4 4.78 1.31 >32 >32 23 4 >20 21.5 >32 >32 244 >20 >32 3.98 >32 25 4 >20 >32 6.33 >32 26 4 14.1 >32 >32 >32 27 8 1.250.005 (n = 4) >32 >32 n value in column refers to KOR DRX replicates.

Example 5 Pharmacology and ADMET Properties of Declared Antagonist ProbeCompound

The broader selectivity of compound 1{3} was tested against additionaltargets and basic pharmacological properties were assessed. Compound1{3} was subjected to a binding assay panel of 44 GPCR and othermolecular targets utilizing the resources of the Pyschoactive DrugScreening Program (PDSP) (Table 5). The compound was initially screenedin radioligand binding assays at a constant concentration (10 μM) toidentify possible activity of the compound. Results showing significantactivity in the initial screen were selected for K_(i) determinations.

TABLE 5 GCPR K_(i) (nM) 5ht1a X 5ht1b X 5ht1d   702 5ht1e X 5ht2a X5ht2b 1,922 5ht2c X 5ht3 X 5ht5a X 5ht6 X 5ht7 X Alpha1A X Alpha1B XAlpha1D X Alpha2A X Alpha2B 7,695 Alpha2C   694 Beta1 X Beta2 X Beta3 XBZP Rat Brain Site X D1 X D2 1,346 D3   250 D4 X D5 X DAT * DOR 1,443GabaA X H1   454 H2 3,502 H3 X H4 X KOR   129 M1 X M2 X M3 X M4 X M56,397 MOR 1,585 NET   685 SERT 5,326 Sigma1 X Sigma2 X X, K_(i) > 10,000or missed in primary screen. * Assay results pending.

Overall, the Chemotype I probe 1{3} displayed a slightly cleaner bindingprofile than other antagonist compounds tested (compounds of a differentchemotype), although this probe did possess a rather high affinity forthe D3 receptor (K_(i)=250 nM). This affinity, while not optimal, mayalso encourage its development as a D3 receptor antagonist. Compounds ofthis type have also shown great promise in the treatment of addictivedisorders (Heidbreder & Newman (2010) Ann. N.Y. Acad. Sci. 1187:4-34).The KOR binding affinity (K_(i)=129 nM) was in excellent agreement withthe 120 nM (DISCOVERX) estimate for the IC₅₀ inhibition of KOR responseto dynorphin A.

In addition to profiling the selectivity of this class of KOR ligands,basic pharmacological properties were assessed. Thus, compound 1{3} ofChemotype I was characterized by a variety of screens including aqueoussolubility, PAMPA (Parallel Artificial Membrane Permeability Assay),plasma protein binding, plasma stability, and hepatic microsomestability.

The results of this analysis indicated that compound 1{3} had areasonable solubility of 12.9 μg/mL in 137 mM NaCl, 2.7 mM KCl, 10 mMsodium phosphate dibasic, 2 mM potassium phosphate monobasic, pH 7.4(PBS) at room temperature (23° C.). At acidic pHs, the solubility of thecompound improved dramatically to >90 ug/mL (96.6 μg/ml at pH 5.0 and90.6 μg/ml at pH 6.2). In addition, this compound possessed superiorstability at room temperature in PBS in the absence of any antioxidantsor other protectants (<0.1% DMSO v/v) with 94.42% of the parent compoundremaining after 48 hours of incubation.

PAMPA is used as an in vitro model of passive, transcellularpermeability. An artificial membrane immobilized on a filter is placedbetween a donor and acceptor compartment. At the start of the test, drugis introduced in the donor compartment (at various pH). Following thepermeation period, the concentrations of compound in the donorcompartment and acceptor compartment are measured using UV spectroscopy.In this assay, compound 1{3} had a moderate permeability of 27×10⁻⁶ cm/sat pH 5 that increased to 227×10⁻⁶ cm/s at pH 6.2 and to 757×10⁻⁶ cm/sas the pH rose to 7.4, consistent with loss of protonation and positivecharge, which would improve permeability. This probe exhibited moderatepermeability in the blood brain barrier PAMPA assay (donor and acceptorcompartments both at pH 7.4) of 51×10⁻⁶ cm/s.

Plasma protein binding is a measure of a drug's efficiency to bind tothe proteins within blood plasma. The less bound a drug is, the moreefficiently it can traverse cell membranes or diffuse. Highly plasmaprotein bound rugs are confined to the vascular space, thereby having arelatively low volume of distribution. In contrast, drugs that remainlargely unbound in plasma are generally available for distribution toother organs and tissues. In the instant case, the Chemotype Iantagonist 1{3} was marginally available with 88.46% and 80.07% bound to1 μM and 10 μM mouse plasma protein, respectively, and 93.96% and 88.54%bound to 1 μM and 10 μM human plasma protein, respectively.

Plasma stability is a measure of the stability of small molecules andpeptides in plasma and is an important parameter that can stronglyinfluence the in vivo efficacy of a test compound. Drug candidates areexposed in plasma to enzymatic processes (proteinases, esterases), andthey can undergo intramolecular rearrangement or bind irreversibly(covalently) to proteins. Compound 1{3} showed excellent stability (100%remaining after 3 hours) in both human and mouse plasma.

The microsomal stability assay is commonly used to rank compoundsaccording to their metabolic stability. This assay addresses thepharmacologic question of how long the parent compound will remaincirculating in plasma within the body. Compound 1{3} was found to belong lasting with 22% and 7.3% (for human and mouse microsomes,respectively) remaining after 1 hour.

Compound 1{3} was also screened against the NCI-60 panel of human tumorcell lines. The compound was screened against each cell line in a singledose at 10 μM. No significant inhibition of tumor cell growth wasobserved. The absence of selective cytotoxicity was expected given thatthe compound was developed to target the KOR.

Three of the most potent and selective of the KOR antagonists, NorBNI,GNTI, and JDTic have been recognized for their long acting properties atthe KOR that may be associated with JNK activation. While they havecommon structural features their differences are sufficient to cloud theSAR that underlies this long acting physiological behavior that can beblocked by reversible nonselective opioid antagonists. It is thereforeimportant to determine how this selective, structurally novel chemotypefunctions at the KOR with respect to NorBNI, GNTI, and JDTic. Ofparticular interest is whether the probe is long or short acting at theKOR, and studies of this question in cells and animal models may clarifyas to whether the underlying mechanism for antagonist anti-addictivebehavior requires activation of JNK.

Example 6 Mechanism of Action Analysis of Chemotype I

To determine where in the pathway for GPCR activation of the KOPreceptor compound 1{3} acted, mechanism of action studies were carriedout. Compound 1{3} and the isosteric analog compound 1{1}, lacking the4-methyl substitution and the nitrogen heteroatom of the pyridine group,were compared, with Nor-BNI as a control. As evidenced by the datapresented in Table 6, compound 1{3} was found to be a potent inhibitorof the ³⁵S-GTPγS coupling, indicating a direct functional effect onG-coupling. Potency was comparable to the β-arrestin translocation HCSassay.

TABLE 6 Ligand IC₅₀, nM Imax (% NBNI) Nor-BNI 4.64 ± 0.83 101 ± 1 Compound 1{1} 95.7 ± 48  122 ± 5^(a) Compound 1{3}  434.2 ± 154.1^(a)117 ± 5^(a) n ≧ 3 curves performed in duplicate. Ligand vs. NBNI: ^(a)p< 0.05; t-test n ≧ 3.

In addition, compound 1{3} was observed to be a potent inhibitor of thedownstream ERK 1/2 activation pathway (Table 7), the potency of whichwas comparable to the β-arrestin translocation HCS assay.

TABLE 7 Ligand IC₅₀, nM Imax (% NBNI) Nor-BNI 4.46 ± 0.64  100 ± 0.3Compound 1{1}  65 ± 9.2^(a) 101 ± 3  Compound 1{3} 330.0 ± 29.4^(a,b) 90± 2^(a) n ≧ 3 curves performed in replicates of 6. Ligand vs. NBNI:^(a)p < 0.001; Ligand vs. Compound 1{3}: ^(b)p < 0.001, t-test.

Interestingly, it appeared that when KOR β-arrestin potencies wereenhanced, the G protein coupling and ERK activation potencies werediminished. This could be an example of functional selectivity.

Example 7 SAR Expansion and Optimization of Chemotype II

Optimization of Chemotype II compound was also carried out. The modularsynthetic routes to this class of compounds, summarized in Scheme 3,permitted the rapid exploration of structure-activity relationships.This Chemotype could be readily assembled from commercially-available oreasily-accessed fragments, thus allowing the introduction of newfunctional groups or core modifications.

The HTS screening campaign for the triazole-based Chemotype IIoriginally uncovered four compounds with potencies around 2 μM and oneexample at 6.7 μM (Tables 8 and 9, entries 1 and 3-6). While a number ofChemotype II analogues were commercially available, limited substitutionon the phenyl ring and no available thiophene-containing analogues ledto the adoption of an entirely synthetic approach based on precedentedchemistry. Beginning with the appropriate isothiocyanate and 2-picolynylhydrazide, the 1,2,4-triazole-3-thione scaffolds were synthesized in twosteps with excellent yields (77-82% overall yields) withoutchromatographic separations (Burbuliene, et al. (2009) ARKIVOC 281-289).

By way of illustration, compound 2{9} was synthesized as follows.

N-(Thiophen-2-ylmethyl)-2-picolinoylhydrazine carbothioamide

2-Picolynyl hydrazide (883 mg, 6.44 mmol) and thiophene isothiocyanate(1,000 mg, 6.44 mmol) in MeCN (20 mL) were stirred for 16 hours at roomtemperature. The reaction mixture was filtered, the precipitate washedwith additional MeCN (3×10 mL) and dried under vacuum to afford thethioamide as an off-white solid (1,642 mg, 5.62 mmol, 87% yield), whichwas used without further purification. Mp 175-178° C.; ¹H NMR (DMSO-d6)δ 4.84 (d, J=6.0 Hz, 2H), 6.93 (m, 1H), 7.00 (m, 1H), 7.36 (dd, J=1.2,4.8 Hz, 1H), 7.63 (m, 1H), 8.03 (m, 2H), 8.56 (br s, 1H), 8.66 (d, J=4.8Hz, 1H), 9.50 (br s, 1H), 10.60 (s, 1H); ¹³C NMR (DMSO-d6) δ d 122.5,124.9, 125.8, 126.2, 126.9, 137.6, 148.4; u 42.1, 141.9, 149.3, 181.4,198.3; IR (neat) 3141, 1672, 1527, 1499, 1466 cm⁻¹; HRMS (ESI) m/z calcdfor C₁₂H₁₃N₄OS₂ ([M+H]⁺), 293.0531, found 293.0516.

4-(Thiophene-2-ylmethyl)-3-(pyridin-2-yl)-1H-1,2,4-triazole-5(4H)-thione

To a slurry of the above thioamide (602 mg, 2.06 mmol) in water (25 mL)was added NaOH (4.00 g, 100 mmol). The reaction was heated at reflux for2 hours; the starting thioamide dissolving promptly upon warming. Thereaction was cooled to room temperature, diluted with aqueous HCl (1 N,20 mL) and acidified to pH=6 with concentrated HCl. The solidprecipitate was filtered, washed with water (2×15 mL) and dried undervacuum to afford the thiophene thione as a white solid (530 mg, 1.93mmol, 94% yield), which was used without further purification. Mp229-231° C.; ¹H NMR (DMSO-d6) δ 6.02 (s, 2 H), 6.87 (dd, J=3.2, 4.8 Hz,1H), 7.09 (d, J=2.4 Hz, 1H), 7.34 (dd, J=0.8, 5.2 Hz, 1H), 7.59 (q,J=4.4 Hz, 1H), 7.99 (d, J=4.4 Hz, 2H), 8.80 (d, J=4.8 Hz, 1H), 14.2 (brs, 1H), 10.60 (s, 1H); ¹³C NMR (DMSO-d6) δ d 122.8, 125.5, 126.3, 126.5,128.1, 138.0, 149.0; u 42.3, 137.8, 145.5, 147.8, 168.3; IR (neat) 3019,2896, 1584, 1549, 1501, 1462 cm⁻¹; HRMS (ESI) m/z calcd for C₁₂H₁₁N₄S₂([M+H]⁺), 275.0475, found 275.0412.

2-(5-((3,4-Dichlorobenzyl)thio)-4-(thiophen-2-ylmethyl)-4H-1,2,4-triazol-3-yl)pyridine2{9}

The thiophene thione (65 mg, 0.24 mmol), K₂CO₃ (66 mg, 0.48 mmol) and2,4-dichlorobenzyl chloride (56 mg, 0.28 mmol) were combined in acetone(3 mL) and stirred in a sealed vial for 15 hours. The solvent wasremoved and the residue washed with CH₂Cl₂ (2×3 mL) then filtered. Thecombined filtrates were concentrated and purified by silicachromatography to afford the triazole product as an off-white solid (92mg, 0.21 mmol, 90% yield). Mp 162-163° C.; R_(f)=0.24 (1:1hexanes:EtOAc); ¹H NMR (CDCl₃) δ 4.42 (s, 2H), 5.93 (s, 2H), 6.85 (dd,J=3.6, 4.8 Hz, 1H), 6.98 (d, J=2.4 Hz, 1H), 7.15 (dd, J=0.8, 4.8 Hz,1H), 7.23 (dd, J=1.6, 8.0 Hz, 1H), 7.33 (m, 2H), 7.48 (d, J=2.0 Hz, 1H),7.80 (dt, J=1.6, 8.0 Hz, 1H), 8.29 (d, J=8.0 Hz, 1H), 8.66 (d, J=3.6 Hz,1H); ¹³C NMR (CDCl₃) δ d 123.2, 124.3, 126.4, 126.5, 127.7, 128.6,130.6, 131.0, 137.1, 148.6; u 36.6, 43.8, 131.9, 132.6, 137.7, 147.6(×2), 152.0, 152.5; IR (neat) 3052, 1589, 1568, 1463 cm⁻¹; HRMS (ESI)m/z calcd for C₁₉H₁₅Cl₂N₄S₂ ([M+H]⁺), 433.0115, found 433.0108.

The subsequent coupling with a wide range of benzyl halides proceededsmoothly in acetone facilitated by K₂CO₃ (Dilanyan, et al. (2008) Chem.Heterocycl. Compd. 44:1395-1397) to readily furnish over 75 compounds, aselection of which is shown in Table 8.

TABLE 8

Entry 2 {n} CID SID X R Purity (%) Yield (%) 1  662944 87218751 O2,4-dichloro ND NA 2 44601469 87334048 S 2,4-dichloro >99  76 3  66329087218759 O 4-bromo ND NA 4 44601472 87334045 S 4-bromo >99 53 5  1982054 4260946 O Styryl^(a) ND NA 6  2482316 17388459 O 4-t-butyl ND NA 7 662263  860989 O 3-chloro ND NA 8 44601470 87334039 O 3,4-dichloro >99 77 9 44601475 87334049 S 3,4-dichloro >99  90 10 44620914 87544155 O4-chloro-3- 98 68 trifluoromethyl 11 44620925 87544171 S 4-chloro-3- 9986 trifluoromethyl 12 44601474 87334041 O 4-methyl >99  65 13 4460147387334044 S 4-methyl >99  88 14 44620937 87544143 O4-trifluoromethyl >99  57 15 44620933 87544159 S 4-trifluoromethyl 99 9316 44620923 87544150 O 3,5-difluoro 97 83 17 44620926 87544166 S3,5-difluoro 99 66 18 44620927 87544153 O 2,4,6-trimethyl 99 80 1944620916 87544169 S 2,4,6-trimethyl 99 43 20  2562032 87334040 O4-methoxy >99  97 21 44601471 87334046 S 4-methoxy >99  88 22 4460146687334042 O H >99  70 23 46601467 87334043 S H >99  83 24 4460146887334047 S 2,4-difluoro 93 72 25  1423675 87544141 O 4-nitro >99  48 2644620924 87544157 S 4-nitro 93 60 27 44620930 87544142 O 4-cyano 97 7428 44620938 87544158 S 4-cyano 99 88 29 44620928 87544144 O4-methylcarboxylate >99  60 30 44620922 87544160 S 4-methylcarboxylate99 83 31  4462915 87544145 O 4-acetoxy 94 41 32 44620931 87544149 S4-acetoxy 98 55 33 44620920 87544146 O 4-isopropyl >99  62 34 4462092987544162 S 4-isopropyl 99 78 35 16447357 87544147 O 2-methyl 99 57 3644620912 87544163 S 2-methyl 99 64 37 44620913 87544148 O 3-methoxy 9888 38 44620918 87544164 S 3-methoxy 99 67 39 44620932 87544149 O2-methyl-3-nitro 95 97 40 44620917 87544165 S 2-methyl-3-nitro >99  8141 16447354 87544151 O 2,6-difluoro 99 73 42 44620936 87544167 S2,6-difluoro 98 98 43 44620919 87544152 S 2,3,4-trifluoro 99 88 4444620921 87544168 S 2,3,4-trifluoro >99  80 45 44620935 87544154 O4-fluoro-2- 99 74 trifluoromethyl 46 44620911 87544170 S4-fluoro-2-trifluoromethyl 99 43 47  662723 87544156 O 2-chloro >99  7948 44620934 87544172 S 2-chloro 99 76 ^(a)Styryl side chain in place ofthe substituted benzyl group.

Mindful of classical SAR substitution strategies (Topliss (1977) J. Med.Chem. 20:463-469; Hajduk & Sauer (2008) J. Med. Chem. 51:553-564), theinstant approach was to vary the substitution on the phenyl ring, whichproduced two submicromolar analogues (entries 8 to 11, Table 9).Interestingly, some compounds containing substituted phenyl ringsdisplayed >100% E_(max) values at the highest concentration tested (ascompared to dynorphin A (E_(max)=100%)).

TABLE 9 Average Potency (μM) Entry KOR KOR MOR K_(i) 2{n} E_(max) (%)DRx^(a) HCS^(b) HCS^(b) (nM) 1 ~100 1.86 ± 0.07 0.93 >32 2 ~120 2.27 ±0.46 8.11 >32 3 ~100 1.92 ± 0.05 1.36 >32 4 ~185 1.85 ± 0.11 1.10 >32 5~100 2.22 ± 0.10 1.43 >32 6 ~100 2.01 ± 0.07 0.60 >32 7 ~100 6.76 ± 0.013.63 >32 8 ~140 0.87 ± 0.06 0.347 >32 2 9 ~135 0.73 ± 0.11 0.43 >32 10~122 0.50 0.46 >32 11 ~109 0.43 1.4 >32 3 12 ~150 6.20 ± 1.26 5.21 >3216 13 ~170 3.59 ± 0.30 2.38 >32 14 ~166 2.77 1.25 >32 15 ~149 1.230.6 >32 16 ~104 7.89 21.0 >32 17 ~150 7.20 >17.8 >32 94 18 ~100 15.09.8 >32 19 ND >20 9.5 >32 20 ~150 7.70 ± 1.47 12.4 >32 28 21 ~130 9.62 ±1.32 5.98 >32 7.1 22 E_(max) not reached >17.90 >32 >32 155 23 ~15013.85 ± 1.18  23.5 >32 138 24 ~155 6.74 ± 0.97 5.68 >32 25 ~120 2.482.35 >32 26 ~128 1.24 0.96 >32 27 ~105 6.79 19.0 >32 28 ~142 4.643.5 >32 29 ~125 6.62 18.5 >32 30 ~154 6.14 6.0 >32 31 ND >20 >32 >32 32ND >20 >32 >32 33   165 5.97 2.8 >32 34 ~100 3.66 2.2 >32 2.3 35ND >18.8 >30 >32 123 36 ND >16.0 14.5 >32 68 37 ND >17.6 >32 >32 67 38~100 8.72 12.2 >32 39 ~128 8.21 12.0 >32 40 ~105 5.81 4.2 >32 41ND >20 >32 >32 55 42 ND >19.0 20.9 >32 78 43 ~140 7.00 5.3 >32 44 ~1153.04 1.7 >32 45 ~138 8.27 5.4 >32 46 ~109 4.46 2.25 >32 47 ND 9.0711.6 >32 106 48 ~100 10.15 4.1 >32 ^(a)DISCOVERX β-arrestin PATHHUNTERassay (n = 3). ^(b)High content imaging based β-arrestin translocationassay (n = 2).

Replacement of the pyridyl and furan/thiophene side chains was alsobriefly investigated and the compounds screened in the DISCOVERX KORassay.

The pyridyl side chain appeared to be critical to the potency, howeverthe furan/thiophene was successfully replaced with other aromaticmoieties without detriment to the potency. These limited examplesindicate that the core scaffold can tolerate an expanded range ofdiverse functionality and opens up several additional structuralelements to exploration.

Example 8 Pharmacology and ADMET Properties of Declared Agonist ProbeCompound

As a representative of the Chemotype II series, the MLPCN probe molecule2{8} was subjected to a binding assay panel of 44 GPCR and othermolecular targets by the Pyschoactive Drug Screening Program (PDSP)(Table 10). The compound was initially screened in a radioligand bindingassay at a constant concentration (10 μM) to identify possible activityof the compound. Results showing significant activity in the initialscreen were selected for K_(i) determinations.

TABLE 10 GCPR K_(i) (nM) 5ht1a X 5ht1b X 5ht1d X 5ht1e X 5ht2a 37885ht2b 1237 5ht2c X 5ht3 X 5ht5a 4986 5ht6 X 5ht7 X Alpha1A X Alpha1B XAlpha1D X Alpha2A X Alpha2B 5525 Alpha2C 9601 Beta1 X Beta2 X Beta3 7312BZP Rat Brain Site X D1 1796 D2 X D3 X D4 X D5 7018 DAT X DOR  5351^(a)GabaA X H1 X H2 X H3 X H4 X KOR    2.4^(a) M1 X M2 X M3 X M4 X M5 4418MOR  1900^(a) NET 5870 SERT X Sigma1 X Sigma2 2905 X, K_(i) > 10,000 nMor missed in primary screen. ^(a)Average of separate K_(i)determinations.

Although modestly more potent analogues were subsequently found, thisselectivity data was representative of this series. The test compoundwas found to possess weak binding affinity for 11 nonopioid targets withK_(i) values in the 1 to 10 μM range. In contrast, the binding affinityfor the KOR was at least 500-fold more potent (K_(i)=2.4 nM). Thecompound was found to possess a KOR:DOR selectivity ratio of over1:2,000 and a KOR:MOR selectivity ratio of 1:792 in the secondarybinding assays. However, the binding affinity values did not correlatewith the functional assay data (EC₅₀=870 nM).

In addition to profiling the selectivity of compound 2{8}, the basicphysical properties were also assessed. This analysis indicated that thesolubility of compound 2{8} in aqueous buffer was <0.1 μg/mL at pH 5.0,0.29 μg/mL at pH 6.2, and 0.14 μg/mL at pH 7.4. In the PAMPA screen(Kansy, et al. (1998) J. Med. Chem. 41:1007-1010; donor compartment pH's5.0/6.2/7.4; acceptor compartment pH 7.4), compound 2{8} have excellentpermeability (1793×10⁻⁶ cm/s at pH 5.0; 1921×10⁻⁶ cm/s at pH 6.2; and2089×10⁻⁶ cm/s at pH 7.4). Furthermore, compound 2{8} had almost 3-foldselectivity in the blood brain barrier (BBB-Pe) PAMPA assay (242×10⁻⁶cm/s in in aqueous buffer; donor and acceptor compartments both at pH7.4). This agonist probe was also determined to be highly bound (>99%)to both human and mouse plasma. Moreover, this compound showed excellentstability (100% remaining at 3 hours) in both human and mouse plasma andwas rapidly metabolized in either human or mouse microsomes (0.03%remaining after 1 hour). In addition, the LC₅₀ value of 2{8} towardFa2N-4 immortalized human hepatocytes was >50 μM.

G protein coupling assays using a ³⁵S-GTPγS binding assay wereundertaken to demonstrate single point efficacy for the compound 2{8}.Compound 2{8} was found to be a potent inhibitor of the ³⁵S-GTPγScoupling (EC₅₀=55.2±11.8 nM; E_(max) 101±10%), indicating a directfunctional effect on G-coupling. Potency was comparable to theβ-arrestin translocation HCS assay.

Since agonists can differentially activate GPCRs to engage diversesignaling cascades, a secondary, downstream MAP kinase assay (Erk1/2activation) was used to evaluate agonist activity. Single point efficacycurves revealed a tendency for compound 2{8} to be more efficacious inthis assay (EC₅₀=520±36.8 nM, p<0.001 agonist vs. U69593; E_(max)145±8%, p<0.01 agonist vs. U69593).

Example 9 Analysis of Additional Chemotype II Compounds

Additional triazole analogues were prepared and analyzed in a β-arrestinrecruitment assay. The analogs analogue and their activity are listed inTable 11.

TABLE 11

Entry R¹ R² R³ Potency, EC₅₀ (DRx^(a), μM)  1 2-pyridyl 4-methoxy-phenyl >20   phenyl  2 2-pyridyl 4-methoxy- 4-bromo- >20   phenyl phenyl 3 2-pyridyl 4-methoxy- 3,4-dichloro- 11.80 phenyl phenyl  4 2-pyridyl4-methoxy- 4-chloro-3- >19.7  phenyl triflouoro- methylphenyl  52-pyridyl phenyl phenyl >20    6 2-pyridyl phenyl 4-bromo-  4.06 phenyl 7 2-pyridyl phenyl 3,4-dichloro-  1.24 phenyl  8 2-pyridyl phenyl4-chloro-3-  2.22 triflouoro- methylphenyl  9 2-pyridyl 3-pyridylphenyl >20   10 2-pyridyl 3-pyridyl 4-bromo-  8.51 phenyl 11 2-pyridyl3-pyridyl 3,4-dichloro-  0.76 phenyl 12 2-pyridyl 3-pyridyl 4-chloro-3- 0.50 triflouoro- methylphenyl 13 2-pyridyl 2-chloro- phenyl >20  phenyl 14 2-pyridyl 2-chloro- 4-bromo- 14.23 phenyl phenyl 15 2-pyridyl2-chloro- 3,4- 11.80 phenyl dichloro- phenyl 16 2-pyridyl 2-chloro-4-chloro-3-  4.30 phenyl triflouoro- methylphenyl 17 4-pyridyl 2-furylphenyl >20   18 4-pyridyl 2-furyl 4-bromo- >20   phenyl 19 4-pyridyl2-furyl 3,4-dichloro- >20   phenyl 20 4-pyridyl 2-furyl4-chloro-3- >20   triflouoro- methylphenyl 21 4-bromo- 2-furylphenyl >20   phenyl 22 4-bromo- 2-furyl 4-bromo- 13.48 phenyl phenyl 234-bromo- 2-furyl 3,4-dichloro- 11.00 phenyl phenyl 24 4-bromo- 2-furyl4-chloro-3- 13.28 phenyl triflouoro- methylphenyl ^(a)DISCOVERXβ-arrestin PATHHUNTER assay (n = 4)

While the potency in the β-arrestin recruitment assay for many of thesecompounds was not as good as other compounds described herein,experiments with other members of this chemotype indicate that thesecompounds may have a surprisingly high affinity for the KOR and/ormodulate distinct KOR signaling pathway. Therefore, these compounds, aswell as those listed below, may be of use in modulating the kappa opioidreceptor.

Example 10 Plasma and Brain Concentrations Chemotype II Compounds

Compounds KUIP10051N and KUC104186N (compound 2{41}) were prepared in avehicle composed of 10% DMSO (dimethylsulfoxide) and 10% TWEEN 80 inwater and were administered intraperitoneally (i.p.) at a dose of 10mg/kg (injection volume of 10 μL/mg mouse body weight).

Adult male C57BL/6 mice from Jackson Labs were used (age ˜2 months).Plasma and brains were collected at 30 and minutes from two groups ofmice (n=3). One mouse was excluded from the 30 minute time point forKUIP104186N due to mis-injection. Isolated plasma and whole brainhomogenates were subject to analysis using LC/MS. Concentrations weredetermined from standard curves prepared in the appropriate matrix andare presented in FIG. 1.

1-12. (canceled)
 13. A pharmaceutical composition comprising aneffective amount of a kappa opioid receptor effector, or apharmaceutically acceptable salt thereof, and a physiologicallyacceptable carrier, wherein the kappa opioid receptor effector is anantagonist of Formula I or agonist of Formula II:

wherein X, Y and Z are independently selected from H,H; O; S or NH; R¹is H, a halogen group or a substituted or unsubstituted lower alkyl oralkoxy group; R² is present or absent, and when present is a substituenton one or more ring atoms and is for each ring atom independently H, ahalogen group, or a substituted or unsubstituted lower alkyl or alkoxygroup; X′ is hydrogen or Br; each Z′ is independently C, CH or N; n is 0or 1; R³ is phenyl, substituted phenyl, naphthyl, cycloalkyl, propargylor allyl; and R⁴ is aryl, heteroaryl or cycloalkyl.
 14. Thepharmaceutical composition of claim 13, further comprising a kappaopioid receptor agonist.
 15. The pharmaceutical composition of claim 13,wherein R⁴ is 2-furyl, 2-thiophene or 3-pyridyl.
 16. A method forselectively modulating the activity of kappa opioid receptor comprisingcontacting a kappa opioid receptor with a kappa opioid receptoreffector, or a pharmaceutically acceptable salt thereof, wherein thekappa opioid receptor effector is an antagonist of Formula I or agonistof Formula II:

wherein X, Y and Z are independently selected from H,H; O; S or NH; R¹is H, a halogen group or a substituted or unsubstituted lower alkyl oralkoxy group; R² is present or absent, and when present is a substituenton one or more ring atoms and is for each ring atom independently H, ahalogen group, or a substituted or unsubstituted lower alkyl or alkoxygroup; X′ is hydrogen or Br; each Z′ is independently C, CH or N; n is 0or 1; R³ is phenyl, substituted phenyl, naphthyl, cycloalkyl, propargylor allyl; and R⁴ is aryl, heteroaryl or cycloalkyl.
 17. The method ofclaim 16, wherein R⁴ is 2 furyl, 2-thiophene or 3-pyridyl.
 18. A methodof selectively modulating kappa opioid receptor activity in a patientcomprising administering to a subject in need thereof a kappa opioidreceptor effector, or a pharmaceutically acceptable salt thereof,wherein the kappa opioid receptor effector is an antagonist of Formula Ior agonist of Formula II:

wherein X, Y and Z are independently selected from H,H; O; S or NH; R¹is H, a halogen group or a substituted or unsubstituted lower alkyl oralkoxy group; R² is present or absent, and when present is a substituenton one or more ring atoms and is for each ring atom independently H, ahalogen group, or a substituted or unsubstituted lower alkyl or alkoxygroup; X′ is hydrogen or Br; each Z′ is independently C, CH or N; n is 0or 1; R³ is phenyl, substituted phenyl, naphthyl, cycloalkyl, propargylor allyl; and R⁴ is aryl, heteroaryl or cycloalkyl.
 19. The method ofclaim 18, wherein the subject has an ethanol use disorder, an anxietydisorder, a depressive illness, or schizophrenia.
 20. The method ofclaim 18, wherein the subject has post-traumatic stress disorder. 21.The method of claim 18, wherein R⁴ is 2 furyl, 2-thiophene or 3-pyridyl.